Abnormal cellular proliferation, notably hyperproliferation, is the source of numerous diseases, the most severe one being cancer. In the United States alone approximately 1.5 million people are diagnosed with cancer and 0.5 million die from it each year. The fight against cancer has seen some success but also numerous set-backs. Severe side-effects of anti-cancer drugs and the development of resistant off-spring of cancerous cells are major problems, as is the early and precise localization of tumours and metastasis.
Hyperproliferative cells, such as many cancerous cells, depend on an increased supply of nutrients, growth factors, energy and vitamins. Using the supply route of a vitamin, which is essential for cellular growth and is often in short supply, one might possibly transport drugs to these unwanted cells.
Cobalamin (Cbl), also known as Vitamin B12 and present as cyano-cobalamin (CN-Cbl), hydroxy-cobalamin (HO-Cbl) or aquo-cobalamin (H2O-Cbl), is essential for life and its concentration in the body is very low. Higher organisms including humans have to get the vitamin from their food. The biosynthesis of cobalamin is limited to some prokaryotic organisms, such as anaerobic bacteria. Cobalamin is important for the proper function of the nervous system and is necessary for the proper metabolism of carbohydrates, proteins and fat. Cobalamin is utilized in essential intracellular metabolic pathways. As methyl-cobalamin (Me-Cbl), it functions as a cofactor for methionine synthase. As 5′-deoxyadenosyl-cobalamin (Ado-Cbl), it functions with methylmalonyl-CoA mutase in the rearrangement of methylmalonyl-CoA to succinyl-CoA. A cobalamin deficiency can result in pernicious anemia. Cobalamin is also involved in the reductive conversion of ribonucleotides to deoxyribonucleotides to generate DNA.
In mammals, most of the cellular uptake of cobalamin is regulated by serum transport proteins and by cell membrane receptors. There are two types of cobalamin-binding proteins in plasma: the non-glycosylated protein transcobalamin II (TCII) and the glycosylated proteins transcobalamin I and III (TCI and TCIII), also called R-binder proteins or haptocorrins. TCI and TCIII are immunologically cross-reactive and probably differ only in their carbohydrate composition. TCI is the primary R-binder found in circulation. For simplicity reasons the term TCI will be used when referring to both R-binder proteins TCI and TCIII. Both types of transport proteins (vectors) TCI and TCII circulate in mammalian blood either partly saturated (holo), or partly unsaturated (apo) with cobalamin. A vector-less uptake system for cobalamin with a rather low efficiency in normal cells is also present in mammalian cells (see Sennet, C. and Rosenberg, L. E., Ann. Rev. Biochem. 50, 1053-86 (1981)).
TCII functions in the delivery of plasma cobalamin to all metabolically active cells by receptor mediated endocytosis. It is well known that accelerated cellular proliferation in neoplasia primarily entails increased consumption of cobalamin loaded TCII from circulation by receptor mediated endocytotic uptake. Upregulation in the number of TCII receptors has been widely demonstrated in malignant cell lines to meet the increased metabolic demand of thymidine and methionine production, methylation reactions for DNA synthesis and cellular energetics via mitochondrial metabolism.
The general TCII receptor is present in all tissues while a second and more organ-specific TCII receptor, called megalin, is heavily expressed in kidney proximal tubules and several other absorptive epithelia. After endocytotic internalisation, TCII is degraded in the lysosomes and free cobalamin is transported to the cytoplasm and inside the nuclear membrane, where it is converted into Me-Cbl and Ado-Cbl. These two forms are operating as the active coenzymes of vitamin B12. The essential role of TCII is well established by the observation that inherited inborn lack of TCII leads to megaloblastic anemia, detrimental neurological disorders and death if not treated with excess cobalamin.
Almost all cells are able to generate TCII. Many cells such as hepatocytes, fibroblasts, nervous cells, enterocytes and macrophages synthesize elevated amounts of TCII. It is assumed that the vascular endothelium is the primary source of TCII. Approximately 20-30% of the circulating cobalamin is bound to TCII as holo-TCII. This is the metabolically efficient form that ensures the internalisation of cobalamin in all tissues (see Rothenberg, E. et al., in: Chemistry and Biochemistry of B12, ed. R. Banerjee, New York, N.Y., 1999, pp. 441-473).
TCI is present in blood and plasma as well as in most exocrine secretions and other fluids. It is mainly generated in the foregut tissues, gastric mucosa, salivary and lacrimal glands and secretory epithelium of the inner ear. TCI, unlike TCII, does not seem to deliver its cobalamin primarily for cellular uptake, has a long half-life in the blood, and thus holds more than 75% of circulating cobalamin (and corrin) at any given moment. Almost all TCI circulates as holo-TCI. Its role is not fully understood. It has been proposed to function as a bacteriostatic agent by preventing the supply of all sorts of cobalamins and corrins to microorganisms. It may also stabilise adenosyl-cobalamin and protect if from photolysis. In contrast to TCI, which has a higher concentration than TCII in circulation, the level of TCII can be elevated very quickly by de novo synthesis of apo-TCII in response to incoming cobalamin. TCI is generated rather slowly and can not be stimulated substantially in response to any triggering impact (see Alpers, D. and Russell, G., in: Chemistry and Biochemistry of B12, supra, pp. 411-441).
Until now, the vector-less uptake of cobalamin in mammalian cells has not been considered as an alternative route to supply cobalamin derivatives to hyperproliferative cells. It is undisputed that the physiologically important mechanisms for the uptake of cobalamin by benign mammalian cells requires the vectors TCII and TCI (and intrinsic factor in the digestive tract). However, in vivo and in vitro data show that free cobalamin is also able to traverse the plasma membrane without the involvement of a vector protein. Direct evidence for an additional ability to take up free cobalamin comes from the study of children congenitally and totally deficient in TCII, in whom parenteral administration of free cobalamin resulted in a striking remission of clinical and chemical signs of intracellular cobalamin deficiency (see Hall, C. E. et al., Blood, 53, 251-263 (1979)). In vitro studies showed uptake of free cobalamin in HeLa cells and fibroblasts. In HeLa cells, uptake of free cobalamin is between 1% and 20/of that seen for TCII-bound cobalamin. With human fibroblasts, free cobalamin accumulation in a two-hour interval amounts to about 20% of that noted with TCII-bound vitamin. The free vitamin uptake system in human fibroblasts has been studied in some detail by Berliner and Rosenberg (Berliner, N. and Rosenberg, L. E., Metabolism, 30, 230-236 (1981)). Uptake of free CN-[57Co]-Cbl has been established as a biphasic system: The initial uptake component is rapid, saturable and specifically inhibited by excess unlabelled CN-Cbl and OH-Cbl, and complete within 30 min. The second uptake component is slower, linear with time and not inhibited by excess unlabelled cobalamin, and does not plateau even after 8 h, suggesting the characteristic attributes of a non-specific process. The initial mode of uptake has properties of a protein mediated highly specific membrane traversation; it is sensitive to sulfhydryl reagents and markedly inhibited by cycloheximide (Sennet, C. and Rosenberg, L. E., Ann. Rev. Biochem. 50, 1053-86 (1981)). These properties are consistent with the presence of a protein-mediated, facilitated uptake system of free cobalamin in mammals.
It is well established that many bacteria and all eucaryotic protists are auxotrophic for vitamin B12 and able to bind it with higher affinity than mammalian intrinsic factor, TCI and TCII. Bacterial and protozoan B12-binding proteins are vector-less operating cell surface proteins able to bind a wide variety of corrins (including true cobalamin) with high avidity. Therefore, the detection of bacterial infections in the context of a whole body image, following the application of a radio-labelled cobalamin derivative, was no surprise (Collins, D. A. et al., Mayo Clin. Proc. 75, 568-580 (2000)). The development of hyperproliferative forms of mammalian cells may well entail the development by multistep cancerogenesis of more efficient forms of the already present vector-less cobalamin uptake system.
Approaches have been published and patented to use cobalamin as carrier for a broad variety of biologically active agents, including radioactive metal isotopes (see Collins, D. A., U.S. Pat. Appl. No. 2003/0144198). The results obtained in animals and humans, when using radio-labelled cobalamin derivatives, showed labelling of tumour tissues, but also a strong accumulation of radioactivity in healthy tissues, such as kidney and liver. Therefore, imaging and radiotherapy are far from being optimal. The potential for major damages to some healthy parts of the body limits the applications described so far.
There is an obvious need for compounds, compositions and methods to administer diagnostic and therapeutic cobalamin derivatives to rapidly proliferating cells in higher concentrations compared to normal cells. It is the objective of the present invention to provide new methods to identify, synthesise, characterise and apply cobalamin derivatives with higher specificity for cells with abnormally high proliferation, while avoiding the development of resistant cellular off-spring.